Production of primmorphs from disassociated cells of sponges and corals

ABSTRACT

This invention relates to the establishment of a novel method of culturing sponge cells, coral cells and cells from other invertebrates in vitro. The cells cultured in vitro, which can be cultured as units similar to aggregates, are referred to as primmorphs. The method makes the following methods possible for the first time, using cells/aggregates/primmorphs from sponges, corals and other invertebrates: 
     Methods 
     (i) of preparing substances which modulate proliferation and DNA synthesis, 
     (ii) of identifying/detecting environmentally harmful substances, 
     (iii) of culturing bacteria and other micro-organisms, 
     (iv) of preparing asexual reproductive bodies that can be used in aquaculture for growing corresponding organisms, 
     (v) of preparing cell libraries, 
     (vi) of optimising the nutritional requirements of the cells/aggregates/primmorphs, and 
     (vii) of identifying substances which modulate telomerase activity in cells/aggregates/primmorphs.

DESCRIPTION 1. Introduction

This invention relates to the establishment of the first and thereforenovel method of culturing sponge cells, coral cells and cells from otherinvertebrates in vitro. The cells cultured in vitro, which can becultured as units similar to aggregates, are referred to as primmorphs.A method is thus available which makes it possible for the first time,using cells/aggregates/primmorphs from sponges, corals and otherinvertebrates, to introduce methods: (i) of preparing substances whichmodulate proliferation and DNA synthesis, (ii) of identifying/detectingenvironmentally harmful substances, (iii) of culturing bacteria andother micro-organisms, (iv) of preparing asexual reproductive bodiesthat can be used in aquaculture for growing corresponding organisms, (v)of preparing cell libraries, (vi) of optimising the nutritionalrequirements of the cells/aggregates/primmorphs, and (vii) ofidentifying substances which modulate telomerase activity incells/aggregates/primmorphs.

The phylum Porifera (sponges), together with the other metazoan phylahas a monophyletic origin, [Müller W. E. G. (1995) Naturwissenschaften82, 321-329]. A fundamental autapomorphic feature of the metazoa,including the Porifera, is, for example, the presence of the receptortyrosine kinase, which can only be found in metazoa [Müller W. E. G.,Schäcke H. (1996) Prog. Molec. Subcell. Biol. 17, 183-208].

Within the metazoa, the Porifera exhibit a plesiomorphic feature whichcan be found in no other higher phylum of metazoa; all (or almost all)their cells have a high level of telomerase activity [Koziol C.,Borojevic R., Steffen R., Müller W. E. G. (1998) Mech. Ageing Develop.100, 107-120]. In principle, this is an indication that sponge cellscannot be subdivided into gametes or somatic cells [Müller W. E. G.(1998a) Progr. Molec. Subcell. Biol. 19, 98-132; Müller W. E. G. (1998b)Naturwiss. 85:11-25]. In higher metazoa not suffering from tumours,virtually only the gametes are always telomerase-positive, whereas thesomatic cells are telomerase-negative [Lange T. v. (1998) Science279,334-335].

Because of this property that all (or virtually all) sponge cells aretelomerase-positive, it might be presumed that sponge cells areimmortal. So far, however, there has not yet been any report ofneoplastic diseases in sponges [De-Flora S., Bagnasco M., Bennicelli C.,Camoirano A., Bojnemirski A., Kurelec B. (1995). Mutagenesis 10,357-364]. In the very first report on the high telomerase activity insponges, it was shown by our group that, when removed from theirassociated tissue and converted into a dissociated state, the cellsbecome telomerase-negative [Koziol C., Borojevic R., Steffen R., MüllerW. E. G. (1998) Mech. Ageing Develop. 100, 107-120]. Cells in asingle-cell suspension very probably die off through apoptosis [WagnerC., Steffen R., Koziol C., Batel R., Lacorn M., Steinhart H., Simat T.,Müller W. E. G. (1998) Marine Biol. 131, 411-421]. In addition, the factthat sponges have a species-specific blueprint led us to postulate thatsponges are equipped with an apoptosis mechanism to replace a certaingroup of cells at a specific time. This assumption was supported by thefinding that cells in the sponge tissue are induced to effect apoptosisin response to endogenous factors (e.g. the addition of heat-treatedbacteria) and to exogenous factors (cadmium) [Wagner C., Steffen R.,Koziol C., Batel R., Lacorn M., Steinhart H., Simat T., Müller W. E. G.(1998) Marine Biol. 131,411-421].

After it had been demonstrated by our group that sponge cells possesshigh telomerase activity [Koziol C., Borojevic R., Steffen R., Müller W.E. G. (1998) Mech. Ageing Develop. 100, 107-120], it appeared an easilyachievable aim to establish a sponge cell culture. Up to now, however,it has only been possible to keep sponge cells alive, as in the case ofthe species Hymeniacidon heliophila [Pomponi S. A., Willoughby R. (1994)In: R. van Soest, A. A. Balkema (Eds.) Sponges in Time and Space,Rotterdam, Brookfield, pp.395-400], Latrunculia magnifica [Ilan M.,Contini H., Carmeli S., Rinkevich B. (1996) J. Mar. Biotechnol. 4,145-149] and Suberites domuncula [Müller W. E. G., Steffen R., RinkevichB., Matranga V., Kurelec B. (1996) Marine Biol. 125, 165-170]. Thesecells do not, however, proliferate [Ilan M., Contini H., Carmeli S.,Rinkevich B. (1996) J. Mar. Biotechnol. 4, 145-149].

One reason why, when kept in vitro, sponge cells have so far onlyremained in a quiescent state is, for example, that the methods ofestablishing the single-cell culture have been inappropriate, i.e.suitable culture conditions and culture media have not been available[Pomponi S. A., Willoughby R. (1994) In: R. van Soest, A. A. Balkema(Eds.) Sponges in Time and Space, Rotterdam, Brookfield, pp. 395-400;Ilan M., Contini H., Carmeli S., Rinkevich B. (1996) J. Mar. Biotechnol.4, 145-149]. The media used were supplemented with foetal calf/bovineserum [Pomponi S. A., Willoughby R. (1994) In: R. van Soest, A. A.Balkema (Eds.) Sponges in Time and Space, Rotterdam, Brookfield, pp.395-400; Ilan M., Contini H., Carmeli S., Rinkevich B. (1996) J. Mar.Biotechnol. 4, 145-149]. So far, it has been assumed that growth factorsfound in the serum of vertebrates also stimulate the cell growth ofsponges and other invertebrates. This assumption does not, however,appear appropriate; the reason for this is our findings which show thatsponge cells possess receptors on their surface that are activated byligands which are different from those of mammalian receptors in generaland human receptors in particular. Accordingly, it cannot be regarded asvery probable that growth factors which occur in calf/bovine sera willhave an effect on sponge receptors specifically and invertebrates ingeneral. Furthermore, media rich in sera entail the risk ofcontamination with protozoa [Osinga R., Tramper J., Wijffels R. H.(1998) Trends Biotechnol. 16, 130-134].

What is surprising and novel is our finding, which is the subject matterof the present patent application, that in vitro conditions can bedefined which lead to the formation of multicellular aggregates ofsponges, corals and other invertebrates from dissociated single cells.The aggregates have an appearance similar to tissue and can bemaintained in a culture for more than five months. These aggregates arereferred to as primmorphs. In addition, we describe how cells, havingassociated themselves into primmorphs, become telomerase-positive andare capable of DNA synthesis/cell proliferation.

With the successful establishment of an in vitro culture of cells fromsponges, corals and other invertebrates, a new route has been opened upfor introducing methods of the kind specified at the beginning of thisdescription.

2. Description of the Method

2.1. Material

Natural seawater (S9148), penicillin and streptomycin were obtained fromSigma (Deisenhofen; Germany), RNAguard (24,000 units/ml) from Pharmacia(Freiburg; Germany), the “Telomerase Detection Kit” (TRAPeze) from Oncor(Gaithersburg, Md.; USA), “BrdU-labeling and detection kit” fromBoehringer Mannheim (Mannheim; Germany) and SYBR Green I from MolecularProbes (Leiden; Netherlands).

The compositions of Ca²⁺ and Mg²⁺-free artificial seawater [CMFSW] andCMFSW to which ethylene diamine tetra-acetic acid (EDTA) was added[CMFSW-E] are as described earlier [Rottmann M., Schröder H. C., GramzowM., Renneisen K., Kurelec B., Dorn A., Friese U., Müller W. E. G. (1987)EMBO J 6,3939-3944].

2.2. Keeping Sponges and Other Invertebrates

Samples of the marine sponge Suberites domuncula (Porifera,Demospongiae, Hadromerida) were collected in the Northern Adriatic nearRovinj (Croatia) and then kept in tanks in Mainz (Germany) at atemperature of 16° C.

As representatives of further invertebrates, the soft coralsDendronephthya hemprichi (Cnidaria, Anthozoa, Alcyonaria) were obtainedfrom a pet shop and kept like sponges—with the exception of thetemperature (22° C.).

As an example of sponges, corals and other invertebrates, the studiesusing the sponge Suberites domuncula and the soft coral Dendronephthyahemprichi are described here by way of example.

2.3. Dissociation of the Cells and Formation of the Primmorphs

All the materials and solutions/media are used under sterile conditions.Tissue samples [size usually 4 to 5 cm³] from the sponge/soft coral arecut into smaller pieces [approx. 0.5-1 mm³] of tissue with a scalpel inseawater. After this, these pieces are placed in conical 50 ml tubes[e.g. Falcon—catalogue number 2070] filled with CMFSW-E [ratio of tissueto medium usually 1:10. After gentle shaking, e.g. for 30 min. at 16° C.on a rotating shaker, the supernatant is decanted and discarded. Theremaining pieces of tissue are once again mixed with new CMFSW-E in thesame proportions and again gently shaken by rotation. The supernatant isdecanted and discarded.

The pieces of tissue are now mixed with new CMFSW-E and again moved on ashaker, usually for 40 min. The supernatant, which contains the cells,is filtered through a nylon net with a mesh of e.g. 40 μm, and istrapped in a tube. This step of shaking the pieces of tissue in CMFSW-Eand filtering the supernatant through a nylon net is repeated severaltimes. The cell suspensions are mixed together and gently centrifuged inorder to obtain the cells [usually at 500×g for 5 min.]. The cellsuspension is placed in a seawater/antibiotic solution [antibiotic:usually 100 IU penicillin and 100 μg/ml streptomycin]. This step isusually repeated once or several times. The collected cells areharvested by centrifugation. A cell suspension is adjusted to 10⁷cells/ml, and 1 ml thereof is usually placed in 5 ml seawater/antibioticsolution, e.g. in a 60 mm Petri dish [e.g. Falcon—catalogue number3004].

The cells are incubated, usually at 16° C. and are usually transferredto a new Petri dish four times by careful resuspension withseawater/antibiotic solution. This is intended to ensure that the cellsdo not adhere to the dish. After aggregates have formed, these aretransferred every day from the parent Petri dish which is now available,by pipetting into a conical tube [e.g. 15 ml tube (Falcon—cataloguenumber 2096)]. In the tube, the aggregates settle more quickly. Afterabout 10 sec., the supernatant is decanted and the aggregate suspensionis transferred to new Petri dishes. The aggregates are sucked up with apipette and again washed once or several times, using the gravity methoddescribed, and resuspended in the seawater/antibiotic solution. It ispossible to recover new aggregates from the parent Petri dish severaltimes, usually two to five times. The rinsed aggregates remain in thePetri dishes until they have formed a smooth surface. Usually, theseawater/antibiotic solution is exchanged every day; two thirds of thesolution are replaced by a fresh seawater/antibiotic solution.

Culturing under pressure has also proven suitable for promotingproliferation of the cells.

Following this, the aggregates now forming (primmorphs) (diameter about1-3 mm) are transferred to 24-well plates [e.g. Nunclon™(Nunc)—catalogue number 143982] and 1 ml seawater/antibiotic solution isadded. One or two primmorphs are incubated per well.

If the cultures are stored under generally sterile conditions, it ispossible to dispense with the addition of antibiotics to the seawater.

2.4. Incubation of the Primmorphs with BrdU

In order to determine the cell proliferation, the integration of BrdU[5-bromo-2′-deoxy-uridine] into the cellular DNA was measured using the“BrdU-iabeling and detection kit”, in accordance with the manufacturer'sinstructions.

For this purpose, the primmorphs are incubated, usually in 1 ml of theseawater/antibiotic solution, which also contains the BrdU labellingsolution [final dilution: usually 1:1,000 (10 μM of BrdU)]. Theincubation period is usually 12 hours; the incubation itself is carriedout in culture chambers [e.g. the “culture chamber slides(Nunc)”—catalogue number 177453]. Subsequently, the cells aredissociated in CFMSW-E, washed three times in CFMSW-E andfixed/denatured in 70% ethanol [pH 2.0]. After that, the cells areincubated with anti-BrdU mouse monoclonal antibodies and the immunecomplex is made visible with anti-mouse Ig coupled to alkalinephosphatase and with the dye substrate nitro-blue tetrazolium salt. Thecells are analysed under a light-optical microscope.

2.5. Histological Examinations

The primmorphs are fixed in 4% paraformaldehyde/phosphate-bufferedsaline solution [Romeis, B. (1989) Mikroskopische Technik. Munich; Urbanund Schwarzenberg]. After dehydration with ethanol, the primmorphs areembedded in Technovit 8100 [Beckstead J. H. (1985) J. Histochem.Cytochem. 9, 954-958]. Sections 2 μm thick are prepared and stained withZiehl's fuchsin solution [Martoja R., Martoja M. (1967) Initiation auxTechniques de l'Histologie Animale. Prem. Ed. Masson et Cie., Paris].

2.6. Telomerase Approach

Telomerase activity is detected by means of polymerase chain reaction[PCR] using the “Telomerase Detection Kit (TRAPeze)”; details havealready been described earlier (Kim N. W., Piatyszek M. A., Prowse K.R., Harley C. B., West M. D., Ho P. L. C., Coviello G. M., Wright W. E.,Weinrich S. L., Shay J. W. (1994) Science 266:2011-2014; Koziol C.,Borojevic R., Steffen R., Müller W. E. G. (1998) Mech. Ageing Develop.100, 107-120]. The cell extracts added correspond to 5×10³ cellequivalents. The amplification products are quantified by means ofelectrophoresis using a 12.5% non-denatured polyacrylamide gel in a0.5×TBE buffer [performed in accordance with the manufacturer'sinstructions]. The gels are stained with SYBR Green I in order to detectDNA fragments [Molecular Probes (1996) MP 7567 Jul. 16, 1996]. Thesignals are quantified with a GS-525 Molecular Imager (Bio-Rad). Thedegree of telomerase activity is stated in TPG (total product generated)and is calculated as described [Oncor (1996) TRAPeze telomerasedetection kit; catalogue no. S7700-Kit; second edition. Oncor,Gaithersburg, Md.; USA].

2.7. Statistics

The results were analysed for their significance by means of the pairedStudent's t-Test [Sachs L., Angewandte Statistik (Springer, Berlin)(1984)].

3. Detection of the Biochemical and Cytobiological Properties of thePrimmorphs

3.1. Formation of Primmorphs From S. domuncula Cells

Samples of the marine sponge S. domuncula were used to isolate the cells(FIG. 1A). Single cells were obtained by dissociation as describedabove. After the washing steps specified, the protozoa are removed,which tend to adhere to the surface of the plastic culture vessels. Thecells are transferred to a seawater/antibiotic solution. After the totaltreatment/incubation period, usually five days, primmorphs form (FIG.1D) from the cell aggregates (FIGS. 1B and C). The diameter of the cellaggregates after an incubation time of two days is about 100 μm (FIG.1B) and continues to grow steadily in size; after four days, a diameterof 300 μm is usually reached (FIG. 1C). During this period, theaggregates round themselves off. Normally, after a further three to fivedays, primmorphs ranging between about 1 and 2 mm in size form (FIG.1D).

Cross-sections through the primmorphs analysed under a microscope showthat the cells in the interior are surrounded by a covering ofepithelium-like cells several layers thick (FIGS. 1E and F). The cellswhich form from the squamous epithelium of the primmorphs arepinacocytes, as can be inferred from their flattened fusiformextremities with their distinct nuclei [survey: Simpson T. L. (1984) TheCell Biology of Sponges. Springer-Verlag, New York]; the size of thecells fluctuates around 20 to 30 μm. The cells within the primmorphs aremainly spherulous cells. They have a diameter of about 30 to 40 μm andare characterised by large round vacuoles which occupy the greater partof the cells. The other cells can be referred to as amoebocytes andarchaeoeytes and are about 40 μm in size.

The outer appearance of the primmorphs is smooth and almost spherical(FIG. 1D); the histological sections show that the cells in theprimmorphs are well organised into a tissue-like body (FIGS. 1E and F).It is striking that the squamous epithelium is formed by a layer severalcells thick, usually consisting of pinacocytes (FIG. 1F), which is notfound in natural sponges; this layer is delimited by a single-celledepithelium. The organised arrangement of the cells within the primmorphsalso distinguishes them from the aggregates which are formed fromdissociated cells in the presence of homologous aggregation factor[Müller W. E. G. (1982) Intern. Rev. Cytol. 77, 129-181].

In contrast to the asexual reproductive bodies, buds, reduction bodiesand gemmulae formed in vivo [survey: Simpson T. L. (1984) The CellBiology of Sponges. Springer-Verlag, New York], the primmorphs describedhere form in vitro from a suspension of individual cells. As is shownhere, the dissociated cells form tissue-like bodies. The structure offunctional primmorphs from a suspension of individual cells implies thatthis formation is an active process which comprises ejecting dead cellsand cell fragments; this can be seen under a microscope as “ragged”edges round the primmorphs. The formation of a squamous epithelium frompinacocytes also indicates that sponge cells de-differentiate andsubsequently re-differentiate, namely into those cells which are neededto form primmorphs.

The reorganisation of the cells during their reformation into primmorphspresupposes the presence of structures and associated proteins for cellmigration; as an essential structural element for cell migration,collagen has been thoroughly documented in sponges. The existence of theadhesive glycoprotein fibronectin, which, in higher invertebrates andvertebrates, promotes cellular interactions with the extracellularmatrix, including cell migration, has so far been shown for sponges byimmunological cross-reactions with heterologous antibodies and byisolating the putative cDNA for that polypeptide from the sponge Geodiacydonium [Pahler S., Blumbach B., Müller I. M., Müller W. E. G. (1998)J. Exp. Zool.; in print]. The presence of inorphogens in primmorphs mustbe postulated in order to explain the precise arrangement ofproliferating cells in these bodies. Recently, we isolated the cDNAwhich codes for a possible morphogen, namely “morphogenendothelial-monocyte-activating polypeptide”, from the sponge Geodiacydonium (Pahler et al., 1998a).

3.2. Passaging of the Primmorphs

The primmorphs obtained from S. domuncula have so far been kept inculture for more than five months.

The primary primmorphs can be dissociated again into individual cells inCMFSW-E. The suspension of individual cells formed in this way is stillcapable of forming aggregates and subsequently primmorphs again, whichare now called secondary primmorphs. This process occurs when the cellsare transferred to a seawater/antibiotic solution. The kinetics of theformation of primmorphs are usually identical to those which wereobserved for primary primmorphs. Without Ca²⁺, i.e. in CMFSW-E medium,the cells from primary primmorphs adhere weakly, after dissociation, tothe surface of the culture vessels [e.g. Falcon catalogue number 3004].For optimum adhesion, the vessels need to be gently roughened with acover glass or a rubber scraper.

3.3. Level of Telomerase Activity in Cells as a Function of the CultureConditions

As described by us earlier [Koziol et al., 1998 (s. o.)], sponge cellsgo through a transition, after dissociation into individual cells, froma telomerase-positive state to a ielomerase-negative state.

The level of the telomerase activity was determined in the cells whileprimmorphs were forming from a single-cell suspension. The results showthat cells in the natural cell association have a high level oftelomerase activity; a quantitative analysis showed an activity of 8.9TBG units/5×10³ cell equivalents (FIG. 2; lane a). When the telomeraseactivity was determined in cells which were left in a dissociatedindividual-cell state for 14 hours, the enzyme level dropped to 0.9 TBGunits/5×10³ cell equivalents (FIG. 2; lane b). If, however, cells fromprimmorphs [about 10 days after their formation from single cells] wereused for the analysis, the resulting telomerase activity was 4.7 TBGunits/5×10³ cells (FIG. 2; lane c).

These results show that when released from their tissue association,cells lose their telomerase activity. As has now been shown, theindividual cells recover again after the formation of tissue-likebodies, the primmorphs, and become telomerase-positive again.

3.4. Immunocytochemical Detection of BrdU Incorporation in Cells ofPrimmorphs

BrdU labelling and the detection approach were used in order to showthat those cells which have organised themselves into primmorphs regaintheir ability to proliferate. As a measure of proliferation, the cellswere incubated with BrdU for 12 hours. After that, the incorporation ofBrdU into the DNA was determined and quantified by means of an anti-BrdUmonoclonal antibody, as described above. Cells which have incorporatedBrdU into their DNA are characterised by a dark (dark brown) stainedcell nucleus (FIGS. 3B-D). Controls which were not incubated with theprimary antibody against BrdU are not stained (FIG. 3A).

Suspensions of individual cells which were kept in CMFSW-E for one daydid not contain any cells which went through DNA synthesis (Table 1).The percentage of BrdU-positive cells obtained from cell aggregateswhich are formed after one day from a suspension of individual cells inculture is low (6.5%). On the other hand, the number of cellssynthesising DNA/proliferating in primmorplis is high. As summed up inTable 1, the proportion of BrdU-positive cells in primary primmorphs is33.8% and in secondary primmorphs 22.3%. These figures confirm that thecells which are organised into primmorphs undergo DNA synthesis andafterwards regain their capacity for cell division.

3.5. Immunocytochemical Detection of Proliferation in Cells ofPrimmorphs

In order to determine whether the cells do in fact divide after the DNAsynthesis, the same batches were analysed which were also used to detectthe incorporation of BrdU into cells from the primmorphs of S.domuncula. Those cells which were positive for BrdU and which were stillin the binuclear stage after undergoing mitosis were counted.

The results show that no binuclear stages occur in suspensions ofindividual cells or cell aggregates, whereas in primary primmorphs 19.4%are present in binuclear stages, and 13.8% are in binuclear stages insecondary primmorphs (Table 1).

This proves that cells in primmorphs also undergo cell division inaddition to DNA synthesis.

3.6. Association of Bacteria With Cells in Primmorphs

It is known that virtually all species of sponges live in asymbiosis-like relationship with microorganisms [Müller W. E. G., ZahnR. K., Kurelec B., Lucu C., Müller I., Uhlenbruck G. (1981) J.Bacteriol. 145, 548-558]. Not only prokaryotic, but also eukaryoticorganisms are found which live in association with the host (sponge,coral or other invertebrates). The organisms present in the sponges canbe detected by PCR analysis [Althoff K., Schütt C., Steffen R., BatelR., Müller W. E. G. (1998) Marine Biol. 130, 529-536] and/or byanalysing the rRNA in agarose gel. Whereas prokaryotic rRNA contains twomain species, 23S and 16S rRNA, the two main species found in eukaryoticrRNAs are 28S and 18S rRNA.

The analysis of tissue samples after the extraction of the rRNA andsubsequent separation in the agarose gel shows that, in S. domuncula,apart from the two host rRNAs, 28S and 18S rRNA, also prokaryotic rRNAs,23S and 16S rRNA, are found (FIG. 4; lane a). After dissociation andsubsequent production of primmorphs in culture, the prokaryotic rRNAs,23S and 16S rRNA, are usually also detected (FIG. 4; track lane b).

This shows that primmorphs can also contain micro-organisms without thiscausing any “contamination of the culture”.

3.7. Detecting DNA Synthesis and Cell Proliferation in Primmorphs FromCells From the Soft Coral D. hemprichi

In line with the details provided for primmorphs from the sponge S.domuncula, primmorphs from the soft coral D. hemprichi were also grownin culture.

The results show that in suspensions of individual cells or cellaggregates, no or few cells are positive for BrdU insertion, whereasboth in primary primmorphs and in secondary primmorphs a large number ofcells are BrdU-positive, i.e. undergo DNA synthesis (Table 1).

In addition, it was found that in single-cell suspensions or cellaggregates, no binuclear stages occur, whereas in primary primmorphs12.9% binuclear stages and in secondary primmorphs 8.2% binuclear stagesare present. Thus it has also been shown for D. hemprichi that cells inprimmorphs also undergo cell division in addition to DNA synthesis(Table 1).

TABLE 1 Analysis of the cells for DNA synthesis by means of BrdUlabelling and the “detection kit”. The sponge S. domuncula and the softcoral D. hemprichi were used as models. The suspension of individualcells was incubated with BrdU; the nucleotides incorporated were madevisible immunologically by means of anti-BrdU monoclonal antibodies. Thepercentage of BrdU-positive cells and of binuclear stages is shown foreach batch series. The analysis was performed with: (i) dissociatedcells which were kept for 1 day in CMFSW-E, (ii) cell aggregates from asuspension of individual cells after one day in culture in seawater,(iii) primary primmorphs [formation after 10 days] and (iv) secondaryprimmorphs [formed from one-month-old primary primmorphs afterdissociation into individual cells and subsequent formation of secondaryprimmorphs]. 300 cells were counted per batch. Suberites Dendronephthyadomuncula hemprichi Percentage Percentage of of BrdU- BrdU- positiveBinuclear positive Binuclear Cells analysed: cells stages cells stagesSingle cells 0% 0% 0% 0% [dissociated after 24 hours] From aggregates[after 6.5% 0% 6.5% 0% 24 hours] Primary primmorphs 33.8% 19.4% 21.7%12.9% Secondary primmorph 22.3% 13.8% 18.5% 8.2%

4. Description of the Use of the Method

The use of the method of preparing the primmorph culture will bedescribed with reference to the example of S. domuncula. The effectsshown here were also found in primmorphs from D. hemprichi and aretherefore valid for this species in particular and for sponges[Porifera] and corals [and for Cnidaria in general] and then also forinvertebrates in general.

The primary primmorphs were obtained from individual cells from S.domuncula and used for the experiments after 21 days.

4.1. Using Primmorphs to Identify Substances Modulating DNA Synthesisand Proliferation

The effect of phorbol ester on DNA synthesis and cell proliferation iswell documented for vertebrates [Parker P. J., Dekker L. V. (1997)Protein Kinase C. Springer-Verlag, New York]. The (One) target enzyme ofthese substances is Protein Kinase C. In the course of preliminary work,we were able to show that sponges likewise possess this enzyme [KruseM., Gamulin V., Cetkovic H., Pancer Z., Müller I. M., Müller W. E. G.(1996) J. Molec. Evol. 43, 374-383].

For this reason, the influence of a phorbol ester, phorbol 12-inyristate13-acetate (PMA), on the percentage of BrdU-positive cells in primmorphsfrom S. domuncula was determined. The primmorphs were incubated for twodays with different concentrations of PMA. After that, the DNA synthesiswas determined by means of BrdU labelling as described above. Thecontrol, percentage of BrdU-positive cells in primmorphs without thetest substance, was set at 100%. The results, which are collected inFIG. 5, show that in the concentration range from 0.01 to 1 μg PMA/ml,there is a distinct percentage increase in the number of BrdU-positivecells in primmorphs (FIG. 5).

On the basis of these data, it can be concluded that primmorphs are anexcellent system for detecting biologically active substances ininvertebrates, in this case those which have an etfect on DNA synthesis.

In line with the above comments, it was also examined in parallelbatches whether any change in proliferation occurred after incubationwith the agent used here. It was possible to show that, in the rangefrom 0.01 to 1 μg PMA/ml, there was an increase in the proliferationofecells in primmorphs.

4.2. Using Primmorphs to Analyse Environmentally Harmful Substances

Cadmium is an environmental toxin which is frequently found in theenvironment, and especially in an aquatic milieu [Clark R. B. (1997)Marine pollution. Clarendon Press, Oxford]. This heavy metal wasselected to show that the capacity for DNA synthesis declines as aconsequence of the influence of cadmium on the primmorphs.

The concentrations selected were those also encountered in the naturalenvironment, e.g. in the Northern Adriatic. On the basis of publisheddata, cadmium concentrations of between 0.1 ng/ml (south of Rovinj[Istria]) and 0.5 ng/ml (Pula-Siporex [Istria]) were chosen [Mikulic N.(ed.) (1994) Monitoring programme of the Eastern Adriatic Coastal Area(1983-1991). United Nations Environmental Programme, MAP TechnicalReports Ser. 86, p. 2751]; in addition, higher concentrations were alsoused.

The results show that, after cadmium at a concentration of 1 ng/ml andmore has had time to take effect, a decline in the percentage ofBrdU-positive cells can be measured (FIG. 6). It should be emphasisedthat (i) this exposure was unique and (ii) in the environment, thisheavy metal accumulates in animals; the accumulation can reach a factorof 17,500-fold [Müller W. E. G., Batel R., Lacorn M., Steinhart H.,Simat T., Lauenroth S., Hassanein H., Schröder H. C. (1998) Marine Ecol.Progr. Ser., in print].

On the basis of the experimental data documented here and of those fromthe literature regarding the accumulation of heavy metals in aquaticorganisms, it must be assumed that the system of the primmorphs is asensitive indicator of environmental pollution.

4.3. Production of Suberitine, a Toxic Protein, in Primmorphs

The proof that suberitine is formed in S. domuncula specimens has beenprovided in the literature [Cariello L., Zanetti L. (1979) Comp.Biochem. Physiol. 64C: 15-19]. These sponge specimens were caught innature. Suberitine is a toxic protein which also possesses haemolyticproperties [Cariello L., Zanetti L. (1979)].

It has now been demonstrated for the first time—in the presentspecification—that sponge cells can also produce biologically activesubstances in vitro. Primmorphs from S. domuncula were cultured. Thebiological activity was determined in extracts from the primmorphs after0 to 20 days [transfer of the primmorphs to the 24-well plates].Haemolytic activity was chosen as the parameter. Raw extracts wereprepared from the primmorphs in accordance with the details provided byCariello and Zanetti (1979), and the haemolytic activity was determinedby spectrophotometry. 0.1 optical unit is defined here as 1 arbitraryunit, the HU (haemolytic unit). As documented in FIG. 7, primmorphspossess a slight biological activity of 3.5±0.4 HU/mg protein on the dayof the transfer to 24-well plates. After the primmorphs have beencultured for a period of more than 3 days, there is a significantincrease (P<0.001) in biological activity on 6.9±0.7 HU/mg; after anincubation period of 10 days, the biological activity reaches itsmaximum.

On the basis of the experimental data documented here, it must beassumed that primmorphs are excellent producers of biologically activesubstances in vitro.

The biologically active substances are produced by the primmorphs on asmall (1 ml) or large scale (20 l); in addition, production can alsotake place in large bioreactors and likewise in aquaculture.

TABLE 2 Increase in the size of the primmorphs and in their percentageshare of BrdU-positive cells after the addition of homologous cells(from S. domuncula), and also heterologous cells (from Geodia cydonium)which were killed apoptotically by heat shock. The size of theprimmorphs is indicated by the corresponding diameter. The killed cells(1 × 10³ cells per well) were added to the cultures for three days,after which the results were evaluated. 10 primmorphs in each case weremeasured (the averages and also the standard deviations are shown); 300cells per batch were counted for BrdU-positive cells. The sizes beforethe addition of the cells and after incubation were determined (the sameprimmorphs were evaluated). Without addition +S. domuncula +G. cydoniumBrdU- BrdU- BrdU- Size positive Size positive Size positive (μm) (%)(μm) (%) (μm) (%) Prim- 228 ± 39 31.0 329 ± 41 42.9 287 ± 44 53.8 morphsfrom S. domun- cula

4.4. Identification of Nutrients Which Make it Possible to Increase DNASynthesis and/or Cell Proliferation in Primmorphs

It has been explained above that primmorphs undergo DNA synthesis andcell proliferation in seawater/antibiotic solution without the additionof the standard nutrient media, such as serum.

It will now be shown that both the size of the primmorphs and theirpercentage share of BrdU-positive cells can be significantly (P<0.001)increased by the addition both of homologous cells (from S. domuncula),and also of heterologous cells (e.g. from Geodia cydonium) which havebeen killed apoptotically by heat shock (Table 2).

In addition, it was surprisingly discovered that phosphatidyl serine andalso phosphatidylinositol cause the size of the primmorphs to increaseand also lead to a rise in their percentage share of BrdU-positivecells. The following explanation was found for this. The cells killedapoptotically by heat shock (which serve as a source of nutrients)expose phosphatidyl serine and also phosphatidylinositol and otherlipids on the surface of their cells. These apoptotically modifiedcells/membranes are phagocytised by the primmorphs.

This thus proves that both killed cells and pure components, such aslipids or even collagen or other extracellular molecules have a positiveeffect on the size of the cells and their percentage share ofBrdU-positive cells.

4.5. Primmorphs as a Model for Finding Substances Which ModulateTelomerase Activity

It is known that tumour cells possess high telomerase activity [HastieN. D., Dempster M., Dunlop A. G., Thompson A. M., Green D. K., AllshireR. C. (1990) Nature 346, 866-868]. One objective of medical research isto reduce the activity of this enzyme in tumour cells. Because of thefact that sponge cells are telomerase-positive, the model of theprimmorphs is therefore ideally suited for testing substances whichreduce this activity.

In the following table, it is shown that telomerase activity in cellsfrom primmorphs can indeed by modulated. As the agent, we usedantibodies against the integrin protein cloned by us from S. domunculaand prepared recombinantly. As shown in Table 3, these antibodies reducethe telomerase activity in primmorphs after only 2 days in culture.

This thus shows that primmorphs are an excellent model for findingsubstances which modulate telomerase activity.

TABLE 3 Telomerase activity in cells from S. domuncula after theaddition of antibodies (rabbit-polyclonal) to primmorphs. 50 μl ofantibodies per well were added to the batches. The telomerase activityis stated in TPG (total product generated) and standardised to 5 × 10³cell equivalents. Incubation time Telomerase activity Primmorphs (days)Without antibodies With antibodies + 0 4.9 ± 0.6 4.6 ± 0.7 + 1 4.6 ± 0.54.3 ± 0.6 + 3 4.5 ± 0.5 3.9 ± 0.4 + 10  4.8 ± 0.7 1.2 ± 0.1 + 15  4.4 ±0.4 0.8 ± 0.1

4.7. Using the Primmorphs for Culturing in Aquaculture

Primmorphs can be used for culturing larger tissue cultures and forculturing entire corresponding organisms. Use is made of aquaculture forthis purpose. After an incubation period, usually of two months, in“culture chamber slides” [(Nunc)—catalogue number 177453], for example,primmorphs are transferred together with the latter to larger artificialcontainers (such as tanks), or directly into nature (aquatic milieu).

If larger artificial containers (such as tanks) are used, these arefilled with artificial seawater, as in the case of S. domuncula, andblended with the usual trace minerals. Usually once a week, a littleorganic material, such as tuna fish for example, is added to the medium.After an average of one month, the primmorphs have grown intoorganism-like structures 5 mm in size. Longer incubation times lead tofurther growth to the complete organism.

This thus shows that complex organisms can form again from primmorphskept in aquaculture. In this way, farms of sponges, corals and otherinvertebrates can be established, either in artificial containers (suchas tanks) or in nature.

4.8. Preparing Cell Libraries

Before a cell library is established, it is necessary to check whethercells, e.g. from S. domuncula, will survive freezing and subsequentthawing and will then be capable of functioning again.

For this reason, cells were harvested and frozen, either directly aftertheir dissociation from the starting organism, e.g. the sponge S.domuncula, or from primmorphs. To this end, the cells are frozen slowly(usually 1° C./min) in a suitable tube, usually in dimethyl sulphoxide(usually 10% in seawater) or glycerine (usually 20% in seawater). Thenumber of cells is adjusted to about 5×10⁶ cells/ml. Once a temperatureof about −70° C. is reached, the cells are subsequently transferred toliquid nitrogen and can be stored like this for more than six months.

When the cells are thawed again, they can be raised in stages totemperatures above +0° C. Then the cells are transferred to seawater,usually being added drop by drop. When a temperature is reached whichthe cells also usually require in culture, aggregates and primmorphs canbe established again. No major loss of vitality has been found as aresult of the freezing and thawing process. Furthermore, after theformation of aggregates/primmorphs, the cells exhibit both DNA synthesisand proliferation.

This thus shows that cells from sponges, corals and other invertebratescan be deep-frozen. The possibility therefore also exists of shippingcells in a conventional manner (e.g. in dry ice). Cells can thus becultured at a central location, i.e. cell libraries can be established.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Formation of primmorphs from cells of the sponge Suberitesdomuncula. A. a sample of S. domuncula; magnification×1. The cells aredissociated by treatment in CMFSW-E. After two days in culture, cellaggregates form [medium: seawater/antibiotics] B which increase in size[photographed after three to four days] C; ×10 Usually after five days,primmorphs form D; ×10. E and F, cross-sections through a primmorph showthe multicellular epithelium-like position of pinacocytes surroundingthe inner part, which consists of spherulous cells, amoebocytes andarchaeocytes; E: ×20; F: ×45.

FIG. 2. Telomerase activity in cells from S. domuncula. The telomeraseactivity was determined (i) in cells from tissue (lane a), (ii) in asuspension of individual cells [the cells were analysed for 14 hours,](lane b) and (iii) in primmorphs (lane c). Defined quantities ofmaterial, corresponding to 5×10³ cell equivalents, were incubated in theTRAP batch. After PCR amplification, the products were separated in anon-denaturing polyacrylamide gel; the gel was stained with SYBR Green Iin order to make the DNA fragment visible.

FIG. 3. Cells from primmorphs with the aim of detecting DNA synthesisthere. For this purpose, the primmorphs were incubated in BrdU (furtherdetails in the text); in the process, BrdU is incorporated into theDNA—if any DNA synthesis has occurred. The BrdU units incorporated aredetected by means of an antibody reaction with the help of the“BrdU-labeling and detection kit”. These appear as dark patches whichmark the nucleus of the cell. B-D: cells from primmorphs which have beenincubated with BrdU and subsequently treated with the identificationreagent in order to detect BrdU. In FIG. 3D one BrdU-positive cell isshown, indicated by an arrow; one BrdU-negative cell is marked by anarrow head.

FIG. 4. Analysis of tissue samples and primmorphs for rRNA. The materialwas extracted and the RNA subsequently separated in the agarose gel. Inaddition to the two eukaryotic host rRNAs [euc], 28S and 16S rRNA,prokaryotic rRNAs [proc], 23S and 16S rRNA, are also made visible byethidium bromide.

FIG. 5. Influence of the phorbol ester phorbol 12-myristate 13-acetate(PMA) on the percentage share of BrdU-positive cells in primmorphs fromS. domuncula. The primmorphs were incubated for two days with differentconcentrations of PMA. After this, DNA synthesis was determined by meansof BrdU labelling. The control, percentage share of BrdU-positive cellsamong primmorphs without the test substance, was set at 100%. 300 cellswere counted per batch.

FIG. 6. Influence of cadmium, concentrations between 0.1 ng/ml and 100ng/ml were chosen, on the amount of the percentage share ofBrdU-positive cells among primmorphs.

FIG. 7. Production of suberitine, a toxic protein, in primmorphs from S.domuncula. Primmorphs were cultured. After 0 [transfer of the primmorphsto the 24-well plates] to 20 days, primmorphs were removed and testedfor biological activity. For each incubation point, primmorphs wereremoved in rive parallel batches, a raw extract was prepared and testedfor haemolytic activity. The titre, stated in HU (haemolytic units),relates to 1 mg of protein extract. The average values are shown withthe standard deviations.* (P<0.001).

What is claimed is:
 1. A method of preparing and culturing primmorphsfrom sponges and corals, said method comprising the steps of: (1)cutting tissue samples of sponges or corals into pieces of tissue ofapproximately 0.5-1 mm³, (2) transferring said pieces of tissue to avolume of Ca²⁺ and Mg²⁻-free seawater containing ethylene diaminetetra-acetic acid, (3) gently rotating said volume of Ca²⁺ and Mg²⁻-freeseawater containing said pieces of tissue whereby a supernatantcontaining cells is produced, (4) filtering the supernatant containingthe cells through a net, and trapping the cells in suspension in aculture tube, (5) repeating step (4), (6) combining the cell suspensionsof step (5) to yield a combined cell suspension, (7) incubating thecombined cell suspension in a seawater/antibiotic solution comprisingapproximately 100 IU penicillin and 100 μg/ml streptomycin, (8)adjusting the incubated cell suspension to a high cell density ofapproximately 10⁷ cells/ml, and (9) transferring 1 ml of said adjustedcell suspension and approximately 5 ml of said seawater/antibioticsolution to a Petri dish, (10) incubating the Petri dish atapproximately 16° C. for approximately 24 h, whereby cell aggregatesthat have not adhered to the dish are formed, (11) collecting saidformed cell aggregates, and washing them in seawater/antibiotic solutioncomprising settlement by gravity, (12) transferring said formed cellaggregates to a new Petri dish and carefully resuspending the cells inapproximately 5 ml of said seawater/antiobiotic solution, (13) repeatingsteps (10) to (12) approximately 4 times, (14) removing the primmorphsformed from said cell aggregates and culturing them in seawater orseawater/antibiotic solution with added nutrients, thereby ensuring that(a) the cells associated in the resulting primmorph retain their abilityto perform DNA synthesis and/or cell proliferation and (b) an in vitroculture of proliferating cells is achieved.